Apomixis and ploidy barrier suppress pollen-mediated gene flow in field grown transgenic turf and forage grass (Paspalum notatum Flüggé)

Bahiagrass (Paspalum notatum Flüggé) is the predominant forage grass in the southeastern US. The commercially important bahiagrass cultivar ‘Argentine’ is preferred for genetic transformation over sexual diploid cytotypes, since it produces uniform seed progeny through apomixis. Pseudogamous apomictic seed production in Argentine bahiagrass may contribute to transgene confinement. It is characterized by embryo development which is independent of fertilization of the egg cell, but requires fertilization with compatible pollen to produce the endosperm. Pollen-mediated gene transfer from transgenic, glufosinate-resistant apomictic bahiagrass as pollen donor at close proximity (0.5–3.5 m) with non-transgenic sexual or apomictic bahiagrass cultivars as pollen receptors was evaluated under field conditions. Hybridization frequency was evaluated by glufosinate herbicide resistance in >23,300 seedlings derived from open-pollinated (OP) pollen receptor plants. Average gene transfer between transgenic apomictic, tetraploid and sexual diploid bahiagrass was 0.03%. Herbicide-resistant hybrids confirmed by immuno-chromatographic detection of the PAT protein displayed a single copy bar gene identical to the pollen parent. Hybrids resulting from diploid pollen receptors were confirmed as triploids or aneu-triploids with significantly reduced vigor and seed set as compared to the parents. Transmission of transgenes to sexual bahiagrass is severely restricted by the ploidy difference between tetraploid apomicts and diploid sexual bahiagrass. Average gene transfer between transgenic apomictic tetraploid and non-transgenic, apomictic tetraploid bahiagrass was 0.17%, confirming a very low frequency of amphimixis in apomictic bahiagrass cultivars. While not providing complete transgene containment, gene transfer between transgenic apomictic and non-transgenic bahiagrass occurs at a much lower frequency than reported for other cross-pollinating or facultative apomictic grasses.     

Colon Delivery of Budesonide: Evaluation of Chitosan–Chondroitin Sulfate Interpolymer Complex

The present study was aimed at formulating tablets comprising of coating susceptible to microbial enzyme degradation for releasing budesonide in the colon. Tablets prepared by using Avicel® pH 102 as diluent and Eudragit® L100-55 as binder were coated to a weight gain of 10% w/w employing aqueous mixtures containing chitosan (CH) and chondroitin sulfate (CS). The interpolymer complex between CH and CS was characterized using Fourier transform infrared (FTIR) and differential scanning calorimetery (DSC) studies. The tablets were evaluated for release of budesonide through in vitro in vivo studies. Formation of bonds between –COO^? and –OSO ₃ ^? groups of CS and –NH ₃ ⁺ groups of CH was evident in the FTIR spectra of these interpolymer complexed (IPC) films. The DSC thermograms of these films revealed one endothermic transition between 190°C and 205°C, suggesting the formation of new bonds in the IPC. The pH sensitive swelling exhibited by these films was observed to be a function of CH concentration. Tablets coated with aqueous mixtures containing 40:60 or 50:50 ratio of CH/CS totally prevented the release of budesonide in pH 1.2 buffer. The peaks (FTIR) and endothermic transitions (DSC) characteristic of interpolymer complexation were observed to remain unaffected after sequential exposure of the films to pH 1.2 and pH 7.4 buffer IP. This proved the versatility of these IPC films for colon delivery. C _max of 1,168.99 and 1,174.2 ng/mL, respectively, at 12 and 8 h post-oral dosing of tablets coated with 40:60 or 50:50 ratio of CH/CS was observed in rats. The aqueous CH/CS (40:60) coating could provide a facile method for delivering budesonide to the colon.     

TRIM5 Suppresses Cross-Species Transmission of a Primate Immunodeficiency Virus and Selects for Emergence of Resistant Variants in the New Species

Simian immunodeficiency viruses of sooty mangabeys (SIVsm) are the source of multiple, successful cross-species transmissions, having given rise to HIV-2 in humans, SIVmac in rhesus macaques, and SIVstm in stump-tailed macaques. Cellular assays and phylogenetic comparisons indirectly support a role for TRIM5α, the product of the TRIM5 gene, in suppressing interspecies transmission and emergence of retroviruses in nature. Here, we investigate the in vivo role of TRIM5 directly, focusing on transmission of primate immunodeficiency viruses between outbred primate hosts. Specifically, we retrospectively analyzed experimental cross-species transmission of SIVsm in two cohorts of rhesus macaques and found a significant effect of TRIM5 genotype on viral replication levels. The effect was especially pronounced in a cohort of animals infected with SIVsmE543-3, where TRIM5 genotype correlated with approximately 100-fold to 1,000-fold differences in viral replication levels. Surprisingly, transmission occurred even in individuals bearing restrictive TRIM5 genotypes, resulting in attenuation of replication rather than an outright block to infection. In cell-culture assays, the same TRIM5 alleles associated with viral suppression in vivo blocked infectivity of two SIVsm strains, but not the macaque-adapted strain SIVmac239. Adaptations appeared in the viral capsid in animals with restrictive TRIM5 genotypes, and similar adaptations coincide with emergence of SIVmac in captive macaques in the 1970s. Thus, host TRIM5 can suppress viral replication in vivo, exerting selective pressure during the initial stages of cross-species transmission.     

Impact of tumor microenvironment on oncolytic viral therapy

Interactions between tumor cells and their microenvironment have been shown to play a very significant role in the initiation, progression, and invasiveness of cancer. These tumor–stromal interactions are capable of altering the delivery and effectiveness of therapeutics into the tumor and are also known to influence future resistance and re-growth after treatment. Here we review recent advances in the understanding of the tumor microenvironment and its response to oncolytic viral therapy. The multifaceted environmental response to viral therapy can influence viral infection, replication, and propagation within the tumor. Recent studies have unveiled the complicated temporal changes in the tumor vasculature post-oncolytic virus (OV) treatment, and their impact on tumor biology. Similarly, the secreted extracellular matrix in solid tumors can affect both infection and spread of the therapeutic virus. Together, these complex changes in the tumor microenvironment also modulate the activation of the innate antiviral host immune response, leading to quick and efficient viral clearance. In order to combat these detrimental responses, viruses have been combined with pharmacological adjuvants and “armed” with therapeutic genes in order to suppress the pernicious environmental conditions following therapy. In this review we will discuss the impact of the tumor environment on viral therapy and examine some of the recent literature investigating methods of modulating this environment to enhance oncolysis.     

Biochemical effects of sparfloxacin on cell envelope of mycobacteria.

Sparfloxacin, a difluorinated quinolone is a potent anti-mycobacterial agent used in the treatment of mycobacterial infections. We have investigated whether sparfloxacin had other, more subtle effects on mycobacteria besides its interaction with DNA gyrase that could contribute to its therapeutic efficacy. Mycobacterium smegmatis cells grown in media with sub-inhibitory concentration of sparfloxacin were observed to have significant reduction in the biosynthesis of vital macromolecules, as shown by the incorporation of various radiolabelled precursors. The analysis of subcellular distribution of phospholipids of sparfloxacin-treated cells demonstrated an increase in the cell membrane and reduction in the cell wall, suggesting changes in the cell envelope architecture by sparfloxacin. Significant changes were also observed in other chemical constituents of the cell wall, especially in the arabinose and glucosamine contents. Mycolic acids, the major component of mycobacterial cell wall were reduced in the presence of MIC50 of sparfloxacin. There was a decrease in the limiting fluorescence intensity (Fmax) of 1-anilinonaphthalene 8-sulfonate (ANS) indicating alterations in the organization and conformation of mycobacterial cell surface. These results suggest that the mechanism of action of anti-mycobacterial action of sparfloxacin involves mycobacterial cell envelope.     

Isolation and characterization of brassinosteroids from immature seeds of Camellia sinensis (O) Kuntze

Brassinosteroids play an important role in growth and development of plants. They have been reported universally in all the plants. The present study deals with the presence of these compounds in immature tea seeds. Five brassinosteroids, i.e. 6-deoxo-28-norcathasterone, 6-deoxo-28-norteasterone, 3-dehydro-6-deoxo-28-norteasterone, 6-deoxo-28-nortyphasterol and 6-deoxo-28-norcastasterone have been isolated and identified by GC–MS. The identified brassinosteroids and their derivatives are active constituents of late C-6 oxidation pathway, thereby suggesting the biosynthesis of brassinosteroids in tea seeds by late C-6 oxidation pathway.     

Effects of environmental air pollution on endogenous oxidative DNA damage in humans

Epidemiological studies conducted in metropolitan areas have demonstrated that exposure to environmental air pollution is associated with increases in mortality. Carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) are the major source of genotoxic activities of organic mixtures associated with respirable particulate matter, which is a constituent of environmental air pollution. In this study,we wanted to evaluate the relationship between exposure to these genotoxic compounds present in the air and endogenous oxidative DNA damage in three different human populations exposed to varying levels of environmental air pollution. As measures of oxidative DNA damage we have determined 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and cyclic pyrimidopurinone N-1,N² malondialdehyde-2′-deoxyguanosine (M₁dG) by the immunoslot blot assay from lymphocyte DNA of participating individuals. The level of endogenous oxidative DNA damage was significantly increased in individuals exposed to environmental air pollution compared to unexposed individuals from Kosice (8-oxodG adducts) and Sofia (M₁dG adducts). However, there was no significant difference in the level of endogenous oxidative DNA and exposure to environmental air pollution in individuals from Prague (8-oxodG and M₁dG adducts) and Kosice (M₁dG adducts). The average level of M₁dG adducts was significantly lower in unexposed and exposed individuals from Kosice compared to those from Prague and Sofia. The average level of 8-oxodG adducts was significantly higher in unexposed and exposed individuals from Kosice compared to those from Prague. A significant increasing trend according to the interaction of c-PAHs exposure and smoking status was observed in levels of 8-oxodG adducts in individuals from Kosice. However, no other relationship was observed for M₁dG and 8-oxodG adduct levels with regard to the smoking status and c-PAH exposure status of the individuals. The conclusion that can be made from this study is that environmental air pollution may alter the endogenous oxidative DNA damage levels in humans but the effect appears to be related to the country where the individuals reside. Genetic polymorphisms of the genes involved in metabolism and detoxification and also differences in the DNA repair capacity and antioxidant status of the individuals could be possible explanations for the variation observed in the level of endogenous oxidative DNA damage for the different populations.     

Formation of interspecies fusants of <Emphasis Type="Italic">Agaricus bisporus</Emphasis> and <Emphasis Type="Italic">Agaricus bitorquis</Emphasis> mushroom by protoplast fusion

Interspecies fusants are formed between Agaricus bisporus and Agaricus bitorquis by protoplast fusion technique. Protoplasts were isolated and regenerated by using Novozyme 234 lytic enzyme. Twenty slow growing isolates were separated from the protoplast regenerated colonies, which were assumed as homokaryons (putative homokaryons). These twenty isolates were subjected to growth rate, colony morphology and spawn run studies for screening of true homokaryons. Antifungal markers were developed for selection of fusants.